Microscope image of hepatic organoids. The cytoskeleton is shown in green, cellular nuclei in blue, and a specific hepatic enzyme is marked in red. Credit: Università di Padova.
A new study published in Cell Reports describes a mix of proteins that can guide human stem cells towards forming simplified miniature livers. The method can make generating and growing such ‘organoids’ easier and more efficient, helping scientists who use them for testing drugs or for basic research.
The research team, from Italian, British and Chinese institutions, studied the intrinsic ability of stem cells to create three-dimensional structures. “Ninety per cent of biology is studied in two dimensions on Petri dishes, but cells behave differently in three dimensions” says Nicola Elvassore, associate professor at the Università degli Studi di Padova and lead author of the study. “We wanted to find the proteins that cells use to signal to each other how to differentiate and self-organise in three dimensions”. The team started from human-induced pluripotent stem cells, reprogrammed skin or blood cells, that can differentiate in all types of human cells. They cultured them in tiny chambers with the width of a human hair and about 200-micron high – an experimental setup called a ‘microfluidic environment’. There, the researchers were able to accumulate and identify the proteins secreted while stem cells turned into hepatocytes, the cells that make up most of the liver. Such a study would be impossible on a normal Petri dish, where secreted proteins dissolve and get lost in a much larger volume.
The results1 were a surprise. Most of the secreted molecules were extracellular matrix (ECM) proteins, that are typically found outside the cells and provide them with structural support. “We did not expect hepatocytes to produce a lot of such proteins” says Elvassore. Most significantly, the cells cultivated in microfluidic chambers were much more efficient than those from conventional cultures at forming liver organoids. These are 3D structures, up to 1 millimetre in size and comprising up to tens of thousands cells, that can mimic some key properties of the actual organ. Organoids are used for studying the physiology and development of human organs, as well as for modelling how tissues respond to drugs or to infections.
The researchers then wondered whether the ECM proteins they had found in the micro chambers could be fed to cells from a conventional 2D culture (a Petri dish) and help them form more organoids than they would normally do. They found that they could, and that those organoids proved more efficient than conventionally-cultured ones at some key liver functions, such as ammonia detoxification. The researchers were able to define a detailed protocol, with the right proteins to be administered at each stage of cell differentiation in order to boost organoid production. “Not all labs have access to microfluidic techniques,” notes Elvassore, “and our protocol can bring the same benefits to those who use convention culture systems.”
